PROJECT SUMMARY - TR&D PROJECT 2 Tissue and Cellular Elemental Distribution, and Image Correlation This Technology Research and Development Project will develop Scanning X-Ray Fluorescence Microscopy (SFXM) for quantitative views of trace element distributions in cells and tissues. It will use cryogenic specimen preparation and imaging conditions as a gold standard for chemical and structural preservation as well as radiation damage resistance. We will develop a cryo SXFM capability with a hundredfold increase in accessible specimen area for beamline 8-BM of the Advanced Photon Source (APS) at Argonne National Laboratory, and provide support for its operation. This will double the accessible experimental time in cryo SXFM at the APS, divided between our BTRR and other biomedical researchers via no-cost, peer-reviewed General User Proposals to the APS. We will also introduce cryo specimen cross-compatibility with the nanoscale SFXM capabilities of the NIH- purchased Bionanoprobe at the APS, laser-ablation inductively coupled plasma mass spectrometry (LA-ICP- MS; TR&D 1) at Northwestern, photoacoustic microscopy (PAM; TR&D 2) at Northwestern, and correlative cryogenic confocal light microscope (C3LM) at the APS. We will develop and make available PyElements, a software platform to provide an integrated and cross- registered view of quantitative images from all of these modalities. We will incorporate into PyElements a numerical optimization approach to correcting for self-absorption of x-ray fluorescence in thicker specimens, so that one can obtain quantitative information even from thicker specimens in 3D. All of these developments will take place in the context of our Driving Biomedical Projects, with large-area cryo SXFM being of particular importance to Theme A (Metal homeostasis or dysregulation in brain function), Theme C (Metal fluxes controlling reproduction and development), and Theme D (Metal imbalances in metabolic pathology).